Use of thiol compounds in viral inactivation

ABSTRACT

The invention relates to the use of thiol compounds in viral inactivation in a cell-free environment, especially in the blood, blood plasma, blood serum, conserved blood, blood products, cell culture liquids and nutrient media and to the application of said compounds on the surface of plants and animals. Viral inactivation occurs without requiring cell metabolism.

[0001] This is a national phase filing of the Application No.PCT/DE98/02370, which was filed with the Patent Corporation Treaty onAug. 12, 1998, and is entitled to priority of the German PatentApplication 197 35 120.4, filed Aug. 13, 1997.

I. FIELD OF THE INVENTION

[0002] The present invention relates to the use of thiol compounds forthe inactivation of viruses in a cell-free environment, especially inthe blood, blood plasma, blood serum, conserved blood, blood products,cell culture liquids and nutrient media and when applied onto thesurface of plants and animals. In this connection, viral inactivationoccurs without requiring cell metabolism.

II. BACKGROUND OF THE INVENTION

[0003] Methods of viral inactivation, which have been common by now,comprise the irradiation with radiation rich in energy, such as U.V.light and X-rays. In this case, the objective is to damage nucleic acidmolecules in the viral genome to thereby reduce the information contentof the viral genetic information. By preceding staining of the viralgenome using dyes which can penetrate the viral envelope (e.g., neutralred or proflavines), the viral genome can already be influenced byvisible light. The treatment of virus suspensions with formaldehyde,used in viral inactivation to obtain viral vaccines, also leadspredominantly via a destruction of the viral genome to a reduction ofthe infectiosity of these viruses. Thus, said treatment methodsinfluence substantially the genetic material of the virus, they do notinfluence the composition of the viral envelope and the capability ofthe virus to penetrate the host cell.

[0004] Methods which prevent the attachment of the virus to its hostcell—i.e., the first step of the process of infection- require amodification of the viral coat proteins. Such methods are, e.g., theseparation of the outer viral envelope of what is called “envelopedviruses” by the addition of ether or detergents such as Nonidet P40,Triton X100 or sodium dodecyl sulfate (SDS). Many disinfectants whichare used successfully to combat bacteria, such as chlorine compounds,have no influence on the replication of viruses.

[0005] However, there are also chemical agents having both anantibacterial effect and an antiviral effect. For example, formalin andin certain viruses also alcohols (ethanol, isopropanol) have anantiviral effect at high concentrations. However, these compounds canonly be used for the purpose of disinfection. Their use, e.g., for viralinactivation in blood and blood products is not possible, since thesesubstances having a cytotoxic and denaturing effect also destroy theblood proteins and thus prohibit, or make impossible, a further use ofthe derived products and their application in patients. In addition,said substances are not suitable for viral inactivation on the surfaceof plants, since they would kill the plant.

[0006] An essential requirement for the inactivation of viruses whichare located in the direct vicinity of living cells (e.g., on the leavesof planes) or which occur in liquids in contact with living cells, isrepresented by the fact that the virus-inactivating agent has nocytotoxic properties.

[0007] Therefore, it is the object of the present invention to provideagents for viral inactivation in liquids (particularly in the blood,blood plasma, blood serum and blood products, cell culture liquids,nutrient media) and on the surface of plants and animals, whichdistinguish themselves by a low cytotoxic and/or denaturing effect. Incontrast to the use of radiation rich in energy, the target of theantiviral effect is not the viral nucleic acid but the influence ofviral surface proteins to already prevent the virus from entering intothe host cell.

III. SUMMARY OF THE INVENTION

[0008] The invention relates to the use of thiol compounds in viralinactivation in a cell-free environment, especially in the blood, bloodplasma, blood serum, conserved blood, blood products, cell cultureliquids and nutrient media and to the application of said compounds onthe surface of plants and animals. Viral inactivation occurs withoutrequiring cell metabolism.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows the syncytia formation of HIV-infected H9 cells afterpre-incubation of the virions with NAC.

[0010]FIG. 2 shows an EIA test of untreated HBsAg with 50 mM NAC-treatedHBsAg, untreated HBsAg and with 50 mM NAC-treated HBeAg.

[0011]FIG. 3 shows a reduction of the infectiosity of vaccinia virusesfollowing NAC treatment.

[0012]FIG. 4 shows removal of excess NAC by dialysis.

V. DETAILED DESCRIPTION OF THE INVENTION

[0013] It is the object of the present invention to provide agents forviral inactivation in liquids (particularly in the blood, blood plasma,blood serum and blood products, cell culture liquids, nutrient media)and on the surface of plants and animals, which distinguish themselvesby a low cytotoxic and/or denaturing effect. In contrast to the use ofradiation rich in energy, the target of the antiviral effect is not theviral nucleic acid but the influence of viral surface proteins toalready prevent the virus from entering into the host cell. The objectof the invention is achieved by the subject matters defined in theclaims.

[0014] In particular, this object is achieved by using thiol compoundsfor the inactivation of viruses. This invention differs from thetreatment of viral diseases in that the assistance of the metabolicapparatus of the host cell is surprisingly not necessary for viralinactivation. The present invention does not serve the treatment ofviral diseases, i.e., the prevention of virus replication in a livingorganism, but the inactivation of already existing viruses outside theliving organism. Thus, it can also be carried out in cell-freeenvironments (e.g., in liquids or on the surface of plants and animals).In this connection, the successful use of thiol compounds is based onthe destruction of disulfide bridges in viral proteins.

[0015] The expression “thiol compound” is understood to mean chemicalcompounds which are characterized by the presence of a reduced thiolgroup (SH group). Preferred examples of such thiol compounds arecysteine, cysteine derivatives, mercaptoalkanols, such as methanethiol,ethanethiol or mercaptoethanol, dithiocarbamate, thiophenol and2-mercaptoethane sulfonic acid. Examples according to the invention ofcysteine derivatives are N-acetyl cysteine, N-acetyl cysteinederivatives, such as N-acetyl homocysteine, N-acetyl cysteine ethylester or N,S-diacetylcysteine ethyl ester. N-acetyl cysteine (NAC) andits derivatives are particularly preferred.

[0016] The thiol compounds used according to the invention are alsocharacterized by their non-toxicity within the usually employedconcentration range. This adds to their safety for use in viralinactivation in blood, nutrient media, cell culture liquids and bloodproducts as well as their application in natural environments, e.g.,when plants are sprayed and animals are bathed with dissolved thiolcompounds, respectively.

[0017] The expression “disulfide bridges in viral proteins” refers todisulfide bridges which influence the three-dimensional structure andfunction of viral proteins by intramolecular or intermolecular covalentbonds. Correspondingly, the viral proteins, but at least one viralprotein, contains at least one cysteine residue. The viral proteinpreferably contains several cysteine residues, such as the surfaceproteins of the hepatitis B virus (HBsAg) or the human immunodeficiencyvirus (gp120). It is known that disulfide bridges are stabilized by theconformation of the protein. Therefore, the amount of thiol compound tobe used, which is necessary to separate the already existingintramolecular or intermolecular disulfide bridge(s), depends on theaccessibility of the respective disulfide bridge(s). In this connection,concentrations of up to 100 mM, particularly 1-100 mM, of thiolcompound, have proved their worth in the viral inactivation in theblood, blood plasma, blood serum, conserved blood, blood products, cellculture liquids and nutrient media, particularly in the case of N-acetylcysteine. In this case, the addition can be made by admixing the solidto the liquid or by inserting an aqueous dilution up to the desiredconcentration.

[0018] For the application onto plants, seeds, sprouts, and seedlings orsaplings by means of spraying, immersion, washing or germinationconcentrations of up to 100 mM of thiol compound suffice, particularlyin the case of N-acetyl cysteine. Equal concentrations can also be usedfor the external treatment of domestic and useful animals. This can bedone by rubbing in or rubbing off or bathing the animals in an aqueousdilution of the thiol compound.

[0019] The plants are ornamental and useful plants, e.g., grain (oat,rye, barley, corn, wheat, soybean, rice, millet, couscous), vegetables(pod vegetables, root vegetables, cabbage varieties, leek, potatoes),fruit (apples, pears, lemons, oranges), trees (e.g., citrus fruittrees), foliage and flowering plants as well as further monocotyl anddicotyl plants. Plants are also understood to mean hydroponics andvegetable tissue and cell cultures.

[0020] The viruses to be mentioned are, e.g., DNA viruses, such ashepadnaviridae, parvoviridae, papovaviridae, adenoviridae,herpesviridae, poxviridae, iridoviridae, or PEA viruses, such aspicomaviridae, caliciviridae, togaviridae, flaviviridae,paramyxoviridae, orthomyxoviridae, bunyaviridae, arenaviridae,reoviridae, bimaviridae, and RNA retroviruses, such as HIV.

[0021] Plant-pathogenic viruses are, e.g., tobacco mosaic virus,cauliflower mosaic virus, bromomosaic virus, rice necrosis virus orgemini virus.

[0022] The below examples explain the invention in more detail. Thefollowing preparations and examples are given to enable those skilled inthe art to more clearly understand and to practice the presentinvention. The present invention, however, is not limited in scope bythe exemplified embodiments, which are intended as illustrations ofsingle aspects of the invention only, and methods which are functionallyequivalent are within the scope of the invention. Indeed, variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intendedto fall within the scope of the appended claims.

VI. EXAMPLES A. Example 1 Reduction of the Infectiosity of the HumanImmunodeficiency Virus (Lily) by N-acetyl Cysteine (Nac)

[0023] In order to examine whether NAC influences the HIV infection ofcells, HIV-permissive cells (119 cells) were incubated together withHIV-positive cell culture supernatant and the syncytia formation takingplace upon successful infection of the host cell was observed. The H9cell line is a CD4⁺ T-cell line of human origin (Popovic et al., 1984,Science 224:497-500). The HI virions (viral titer of the originalsolution is 10⁷ viruses/ml) were initially pre-incubated together withNAC at room temperature for two hours (final NAC concentration 0.09 M,dissolved in serum-free cell culture medium) and then mixed with cellculture medium in a decadic dilution series (10⁻¹ to 10⁻⁵). The same wasmade analogously with the control without NAC addition. Thereafter, thethus pretreated virions (and the untreated control, respectively) wereadded to the 149 cell culture for two hours to enable the adsorption ofthe viruses. Then, the suspension was washed with NAC-free medium and48-well cell culture plates were covered therewith. Pour parallel assayswere carried out for each dilution series. The giant cell formation wasobserved microscopically over an observation period of 26 days andassessed on a scale from 0 (no syncytia detectable) to 3 (great numberof syncytia). The result of syncytia formation is shown in FIG. 1 by wayof diagram. It can be seen in connection with the untreated control(FIG. 1) that the syncytia formation is delayed when the viral titerdecreases. The degree of syncytia formation is thus directlyproportional to the number of infectious viruses. While with a viraltiter of 10⁵ the syncytia formation starts within four days already, itis delayed by about 2 weeks when the original virus solution has beendiluted 10,000 times. In contrast thereto, the infectious viruses in theoriginal virus solution are drastically reduced after pretreating theviruses with NAC. If the NAC-treated virions were diluted by more than100 times (viral titer <10⁴), it was no longer possible to observesyncytia formation within the observation period of four weeks at all,whereas the first giant cells had formed in the untreated control undercomparable conditions after one week already.

[0024] Thus, an initial treatment of the viruses with NAC resulted in adrastic reduction of the content of infectious viruses in avirus-containing liquid. The experiment described herein differs clearlyfrom the treatment of HIV-infected cells with NAC. When cells aretreated with NAC, NAC interferes with the cellular metabolism as anoxygen radical trap and reduces in this way the number of viral PEAreplication intermediates which are packaged into the nucleocapsid(Roederer et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 4884-4888).However, in the above described experimental assay the viruses werepre-incubated with NAC in the absence of cells and added to the cellsuspension only afterwards, i.e., after the removal of NAC by dilution.Therefore, the decrease in infectiosity must have been due to adestruction of the virions in the cell-free original virus solution andcannot have been caused by the influence of the cellular metabolism. Asa result, it follows from the data shown that the infectiosity ofHIV-positive blood or HIV-positive blood products can also be reduced byanalogous steps, which adds to the safety of conserved blood.

[0025] The delay of giant cell formation, which is shown in FIG. 1, isdue to a reduction of the number of infectious virions—because of acorrelation between viral titer and the number of syncytia. It is in nocase caused by an influence of the metabolism of the host cell. Thereduced capability to form giant cells (FIG. 1) can be quantified astiter exponent n by observing the laws established by Spearmann-Karber.Class limit −5 −4 −3 −2 −1 Test positives p/4 0/4 2/4 1/4 1/4 4/4 Classmidpoint m −4,5 −3,5 −2,5 −1,5 Increase Δp 2  −1   0  3  Product m*Δp−9    3,5 0  −4,5

[0026] With a total number of 4 preparations per dilution series thereis $n = {\frac{\sum{{m \cdot \Delta}\quad p}}{4}.}$

[0027] Thus, nNAC=−2.5 follows for the titer exponent after NACtreatment. Since the dilution stage of the untreated control, wheresyncytia formation could no longer be observed, was not determinedaccurately in this experiment, the maximum value for this preparation,i.e., 10⁻⁶ was assumed as a calculation basis. Correspondingly, thetiter of the untreated control is 10^(−5,5), or less. The titer afterthe NAC treatment is 10^(−2,5). Measured on the basis of the syncytiaformation capability, the NAC treatment resulted in a reduction of theviral titer by at least three decimal exponents.

[0028] This reduction of the infectiosity titer could also be confirmedby means of the p24-Ag test. For this purpose, the cell culture mediumwas renewed four days prior to the end of the experiment and the p24-Agcontent of the cell culture medium was quantified upon termination ofthe experiment (day 26) (TABLE I). The p24-Ag values of the untreatedcontrol were always clearly positive. As compared thereto, the NACtreatment carried out prior to the infection caused a marked reductionof the p24-Ag content in the cell culture medium from an HIV dilutionfactor of {fraction (1/100)} on, which is equal with a reduction in thenumber of infectious virions and thus also a reduction of the viraltiter (TABLE I). TABLE I The p24-Ag content in the cell culture mediumof HIV-infected H9 cells is reduced if the virions are treated with NACprior to the infection Dilution NAC-treated untreated10⁻¹ >2.0 >2.0 >2.0 >2.0 >2.0 >2.0 >2.0 >2.0 10⁻² 0.045 0.038 >2.00.079 >2.0 >2.0 >2.0 >2.0 10⁻³ 0.044 0.047 0.0390.274 >2.0 >2.0 >2.0 >2.0 10⁻⁴ 0.047 0.044 0.134 0.652 >2.0 >2.01.512 >2.0 10⁻⁵ 0.069 0.04 0.044 0.04 0.238 >2.0 >2.0 >2.0

[0029] The difference between the experiment shown herein and theexperimental preparations published many times already consists in thatthe virions had been treated with NAC prior to the infection of the H9cells, i.e., in the absence of the cells, and were added to the cellsonly afterwards. According to the current state of the art it is commonpractice to permanently treat HIV-infected cells with NAC.Correspondingly, the cause of the anti-HIV effect of NAC must be due tothe effect of NAC as a radical trap, which leads to the known proposalof using NAC for the treatment of HIV infections.

[0030] The here presented reduction of the viral titer by at least threedecimal exponents and the reduction of the p24-Ag content even after 26days following initial infection of the cells, without the cells per sehaving been exposed to NAC, rule out a radical trap effect of NAC as acause of the antiviral effect of NAC. The antiviral effect can rather beexplained by a disturbance of the virus adsorption, by the separation ofdisulfide bridges between conserved cysteine residues in the coatprotein gp120 of HIV. Such a mechanism is independent of the infectedcell and is therefore no treatment of a viral infection. It is ratherthe chemical inactivation of a virus.

B. Example 2 Treatment of the Hepatitis B Virus (Hbv) Surface ProteinHbsag with N-acetyl Cysteine (Nac)

[0031] The cell culture supernatant of a HBV-producing cell line(HepG2-2.2.15 cells, Sells et al., 1984, Proc. Natl. Acad. U.S.A.84:1005-1009) was separated from the cells and then the viral antigens(HBsAg, hepatitis-B virus “surface” antigen) of the viral envelope wereidentified by means of an enzyme immunoassay (EIA). In a parallelexperimental assay, the cell culture supernatant was treated with NACdirectly before the EIA was carried out. While the HBeAg (hepatitis-Bvirus “e” antigen; secretable viral antigen which is not part of theviral envelope) was recognized unrestrictedly also in the presence of 50mM NAC by the test system employed (Abbott HBe EIA), detection of theHBsAg was not possible by the test system (Abbott Auszyme*) (FIG. 2).Since as is known the main antigenic domain of HBsAg is stabilized bynumerous disulfide bridges, it can be inferred from this experiment thatNAC effects a change in the three-dimensional structure on the virussurface, which is caused by a separation of disulfide bridges. Thesevery viral surface structures are essential for the attachment of thevirus to its host cell and thus for its infectiosity.

C. Example 3 Reduction of the Infectiosity of Vaccinia Viruses by Nac

[0032] In order to find out whether NAC has an influence on theinfectiosity of vaccinia viruses, an original vaccinia virus solutionwas pre-incubated with increasing amounts of NAC (3, 10, 30 mM) and thenan infection of permissive cells (CV1 cells, monkey kidney cells) wascarried out. Following the removal of the non-adsorbed viruses, thecells were coated with soft agar medium to prevent further spreading ofthe viruses by diffusion. In this connection, it could be shown that theviral titer of the vaccinia virus suspension was reduced by up to 70%(FIG. 3). This reduction of the infectiosity was markedly above therange of variations of the plaque assay (Akkuranz) which was calculatedon the basis of the number of plaques of the untreated sample and was±9%. As shown similarly for HBV and HIV already, NAC also effects areduction of the infectiosity of vaccinia viruses.

[0033] Therefore, it could be shown for three different viruses (HIV,HBV and vaccinia) which belong to three completely different classes ofvirus (retroviruses (RNA genome), hepadnaviruses (DNA genome) and poxviruses (DNA genome)) that thiol compounds (particularly NAC) effect areduction of the infectiosity of these viruses.

D. Example 4 Method of Removing Nac from Liquids after ViralInactivation

[0034] Hence the addition of NAC to virus-containing liquids effects adecrease in the infectiosity of the liquid (shown for HIV, HBV andvaccinia viruses). Therefore, the use of thiol derivatives (such as NAC)results in an inactivation of viruses particularly in the blood,conserved blood, blood plasma, blood serum and blood preparations. Inthis connection, it may be advantageous to remove the thiol derivativesused in viral inactivation after inactivation of the viruses again. Inorder to examine which influence the removal of NAC fromvirus-containing suspensions has on the infectiosity of the viruses,HBV-positive cell culture supernatant was initially incubated withdifferent amounts of NAC in two parallel assays for 15 minutes. Toremove the unbound NAC again, one assay was dialyzed against TNE buffer(10 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM EDTA) at 4° C. overnight,whereas the other assay remained untreated.

[0035] Irrespective of whether or not the sample was dialyzed overnight,the same curve was obtained in either case (FIG. 4). This shows thatexcess amounts of NAC can be removed by dialysis again without theantigenicity and thus infectiosity of the virus suspension increasingagain. Therefore, it can be recorded that the effect which NAC exerts onthe viruses is irreversible and that dialysis is a suitable method ofremoving excess NAC. The result of FIG. 4 also shows that NAC entersinto chemical bonding with HBsAg. Otherwise the effect could be reversedby dialysis. Therefore, the inactivation of viruses by NAC is a chemicalreaction which takes place between the free SH group of the thiolcompound and a disulfide bridge in the viral protein. The presence of acellular metabolic apparatus is not necessary for this purpose.

E. Examples 5 Viral Inactivation by N-acetyl Cysteine on Plants

[0036] In addition to the wide applicability of thiol derivatives forviral inactivation in liquids, inactivation of viruses in their naturalenvironment is also possible.

[0037] Since as is known NAC shows no cytotoxic effects, it can also beused as an extremely environmentally compatible and simultaneouslybiodegradable preparation for combating plant-pathogenic viruses. It isassessed that losses amounting to about 60 billion US $ are caused byplant viruses each year. Very often, plant viruses are transmittedmechanically. The viruses can be further spread inter alia by directcontact of the infected plant with a neighboring plant, by transmissionof the viruses by means of insects, spores, etc., or via alreadyinfected seeds. Irrespective of the kind of transmission, spraying orimmersion of the host plant (e.g., the seeds) with thiol compounds(e.g., NAC) has a prophylactic effect, since the viruses are alreadyinactivated on the plant surface in this way and can no longer penetratethe interior of the host cell. Thiol compounds can thus make a valuablecontribution to viral inactivation in agriculture as well.

[0038] All references cited within the body of the instant specificationare hereby incorporated by reference in their entirety.

What is claimed is:
 1. Use of thiol compounds for the inactivation ofviruses in cell-free environment.
 2. Use according to claim 1 for viralinactivation in cell-free liquids.
 3. Use according to claim 1 or 2,wherein the cell-free liquids are blood, conserved blood, blood plasma,blood serum, blood preparations, cell culture liquids and nutrientmedia.
 4. Use according to any one of claims 1 to 3, wherein the thiolcompound is added to a concentration of up to 100 mM.
 5. Use accordingto claim 1 for viral inactivation on the surface of plants and animals.6. Use according to claim 5, wherein the thiol compound is applied toachieve a surface concentration of up to 100 mM.
 7. Use according to anyone of the preceding claims, wherein the thiol compounds are cysteine,cysteine derivatives, mercapto alcohols, dithiocarbamate, thiophenol or2-mercaptoethane sulfonic acid.
 8. Use according to claim 7, wherein thecysteine derivatives are M-acetyl cysteine, N-acetyl cysteinederivatives, such as N-acetyl homocysteine, N-acetyl cysteine ethylester or N,S-diacetyl cysteine ethyl ester.